I have been using anti-PDGFRa to IF stain fibroblast populations in both skeletal and cardiac muscle. The results can be very inconsistent, with some stainings looking great and others with essentially no visible fibroblasts. Has anyone experienced these issues with this antibody and been able to fix them? I stain frozen sections from tissues that are fixed in 4% PFA.
Dear Connor!
Please You look a t the protocol:
Article Platelet derived growth factor receptor alpha is essential f...
Human Immunostaining and Microscopy
Cardiac tissue was snap frozen in liquid nitrogen and stored at −80°C. Samples were thawed and fixed in freshly made 4% paraformaldehyde (PFA) at 4°C overnight, paraffin embedded, and sectioned. Five-micrometer sections were permeabilized in 0.5% Triton X-100 for 30 min, blocked in 0.1% Triton X-100 + 1% BSA + 1.5% normal donkey serum in Dulbecco's phosphate-buffered saline (DPBS) for 1 h, and incubated in primary antibodies diluted in block at 4°C overnight. The following antibodies were used for fluorescent immunohistochemistry (IHC) on human sections: PDGFRα (R&D Systems, 1:20, AF-307-SP), Vimentin (Abcam, 1:100, ab-45939), and α-smooth muscle actin (αSMA; eBioscience, 1:100, 53-9760-80). When necessary, secondary antibodies from Thermo Fisher Scientific were used at a 1:500 concentration for 1 h at room temperature. Nuclei were stained with DAPI (Roche, 10-236-276-001). Tissue sections were incubated in 1X TrueBlack Lipofuscin Autofluorescence Quencher (Biotium, 23007) in 70% ethanol for 30 s and washed in DPBS before mounting. A Zeiss Axiovert 200 microscope equipped with an Olympus DP71 camera was used for imaging.
Article Blockade of platelet-derived growth factor receptor-β, not r...
Fibroblast isolation
Mouse lung fibroblasts were obtained according to a previously reported method [13]. The lungs were harvested from untreated mice and minced with scissors. Minced lungs were digested with 0.5% trypsin. Harvested cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL, Rockville, MD) for several days. Proliferating cells (doubling time in 10% FBS: 15 to 18 hours) were used as lung fibroblasts at five to 10 passages. These fibroblasts were positively stained with anti-vimentin and α-smooth muscle actin antibodies, which indicate a myofibroblast phenotype, and negatively stained with an anti-cytokeratin antibody, which indicates an epithelial cell phenotype.
On day 14 of the BLM treatment, harvested lungs were fixed in 10% buffered formalin and embedded in paraffin. Lung sections (thickness of 3 to 4 μm) were deparaffinized and rehydrated. Antigens were retrieved by heating the sections in citrate buffer. Endogenous peroxidase activity was neutralized with 0.3% H2O2. Staining was performed using the VECTASATIN universal quick kit (Vector Laboratories, Burlingame, CA) following the manufacturer’s instructions.
Regarding the detection of PDGFR expression in lung tissue sections, immunohistochemistry was performed as described above. As the primary antibodies, a 1:100 dilution of a rabbit anti-PDGFR-α antibody (Cell Signaling Technology) and a 1:100 dilution of a rabbit anti-PDGFR-β antibody (Cell Signaling Technology) were used.
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